Objective-Degradation of extracellular matrix plays an important role in growth and destabilization of atherosclerotic plaques.Cystatin C, inhibitor of the collagen-and elastin-degrading cysteine proteases of the cathepsin family, is produced by virtually all cell types. It is present in the normal artery wall but severely reduced in human atherosclerotic lesions. Methods and Results-To determine the functional role of cystatin C in atherosclerosis, we crossed cystatin C-deficient (cysC Ϫ/Ϫ ) mice with apolipoprotein E-deficient (apoE Ϫ/Ϫ ) mice. After 25 weeks of atherogenic diet, mice lacking apoE and cystatin C (cysC Ϫ/Ϫ apoE Ϫ/Ϫ ) had larger subvalvular plaques compared with cysC ϩ/ϩ apoE Ϫ/Ϫ mice (766 000Ϯ20 000 m 2 per section versus 662 000Ϯ19 000 m 2 per section; Pϭ0.001), suggesting an atheroprotective role of cystatin C. The plaques from cysC Ϫ/Ϫ apoE Ϫ/Ϫ mice were characterized by increased total macrophage content. To determine which cellular source is important for the antiatherosclerotic effect of cystatin C, we performed bone marrow transplantations. ApoE Ϫ/Ϫ mice were transplanted with either cysC Ϫ/Ϫ apoE Ϫ/Ϫ or cysC ϩ/ϩ apoE Ϫ/Ϫ bone marrow. No significant differences in plaque area, macrophage, collagen, or lipid content of subvalvular lesions between the 2 groups were detected. Conclusions-The result suggests that the protective role of cystatin C in atherosclerosis is dependent primarily on its expression in nonhematopoietic cell types.

ObjectiveIL-25 has been implicated in the initiation of type 2 immunity and in the protection against autoimmune inflammatory diseases. Recent studies have identified the novel innate lymphoid type 2 cells (ILC2s) as an IL-25 target cell population. The purpose of this study was to evaluate if IL-25 has any influence on atherosclerosis development in mice.Methods and ResultsAdministration of 1 μg IL-25 per day for one week to atherosclerosis-prone apolipoprotein (apo)E deficient mice, had limited effect on the frequency of T cell populations, but resulted in a large expansion of ILC2s in the spleen. The expansion was accompanied by increased levels of anti-phosphorylcholine (PC) natural IgM antibodies in plasma and elevated levels of IL-5 in plasma and spleen. Transfer of ILC2s to apoE deficient mice elevated the natural antibody-producing B1a cell population in the spleen. Treatment of apoE/Rag-1 deficient mice with IL-25 was also associated with extensive expansion of splenic ILC2s and increased plasma IL-5, suggesting ILC2s to be the source of IL-5. Administration of IL-25 in IL-5 deficient mice resulted in an expanded ILC2 population, but did not stimulate generation of anti-PC IgM, indicating that IL-5 is not required for ILC2 expansion but for the downstream production of natural antibodies. Additionally, administration of 1 μg IL-25 per day for 4 weeks in apoE deficient mice reduced atherosclerosis in the aorta both during initiation and progression of the disease.ConclusionsThe present findings demonstrate that IL-25 has a protective role in atherosclerosis mediated by innate responses, including ILC2 expansion, increased IL-5 secretion, B1a expansion and natural anti-PC IgM generation, rather than adaptive Th2 responses.

Objective of the StudyDiabetic patients have a much more widespread and aggressive form of atherosclerosis and therefore, higher risk for myocardial infarction, peripheral vascular disease and stroke, but the molecular mechanisms leading to accelerated damage are still unclear. Recently, we showed that hyperglycemia activates the transcription factor NFAT in the arterial wall, inducing the expression of the pro-atherosclerotic protein osteopontin. Here we investigate whether NFAT activation may be a link between diabetes and atherogenesis.Methodology and Principal FindingsStreptozotocin (STZ)-induced diabetes in apolipoprotein E−/− mice resulted in 2.2 fold increased aortic atherosclerosis and enhanced pro-inflammatory burden, as evidenced by elevated blood monocytes, endothelial activation- and inflammatory markers in aorta, and pro-inflammatory cytokines in plasma. In vivo treatment with the NFAT blocker A-285222 for 4 weeks completely inhibited the diabetes-induced aggravation of atherosclerosis, having no effect in non-diabetic mice. STZ-treated mice exhibited hyperglycemia and higher plasma cholesterol and triglycerides, but these were unaffected by A-285222. NFAT-dependent transcriptional activity was examined in aorta, spleen, thymus, brain, heart, liver and kidney, but only augmented in the aorta of diabetic mice. A-285222 completely blocked this diabetes-driven NFAT activation, but had no impact on the other organs or on splenocyte proliferation or cytokine secretion, ruling out systemic immunosuppression as the mechanism behind reduced atherosclerosis. Instead, NFAT inhibition effectively reduced IL-6, osteopontin, monocyte chemotactic protein 1, intercellular adhesion molecule 1, CD68 and tissue factor expression in the arterial wall and lowered plasma IL-6 in diabetic mice.ConclusionsTargeting NFAT signaling may be a novel and attractive approach for the treatment of diabetic macrovascular complications.

Objectives. Accumulation and subsequent oxidation of LDL in the arterial wall are considered as key events in the development of atherosclerosis. We have investigated the possibility that LDL oxidation results in release of aldehydes that modify surrounding matrix proteins and that this may target immune responses against the plaque extracellular matrix and modulate the disease progression.Results. Using custom-made ELISAs we demonstrate that human plasma contains autoantibodies against aldehyde-modified fibronectin (FN) and to a lesser extent also other extracellular matrix proteins including collagen type I, type III, and tenascin-C. Immunohistochemistry and western blot analysis showed that aldehyde-modified FN is present in human atherosclerotic plaques and that aldehydes generated by oxidation of LDL formed adducts with FN in vitro. We also demonstrate that aldehyde-modification of FN results in a loss of its ability to promote basal secretion of cytokines and growth factors from cultured macrophages without affecting the ability of the cells to respond to stimulation with LPS. A prospective clinical study demonstrated that subjects that subsequently developed acute myocardial infarction or sudden cardiac death had lower baseline levels of autoantibodies against aldehyde-modified FN than matched controls.Conclusions. These observations demonstrate that oxidation of LDL in the arterial wall may lead to aldehyde-modification of surrounding extracellular matrix proteins and that these modifications may affect macrophage function and activate autoimmune responses of pathophysiological importance for the development of atherosclerosis.

Background. Diabetes is associated with increased cardiovascular disease, but the underlying cellular and molecular mechanisms are poorly understood. One proposed mechanism is that diabetes aggravates atherosclerosis by enhancing plaque inflammation. The Akita mouse has recently been adopted as a relevant model for microvascular complications of diabetes. Here we investigate the development of atherosclerosis and inflammation in vessels of Akita mice on LDLr −/− background. Methods and Results. Akita-LDLr −/− and LDLr −/− mice were fed high-fat diet from 6 to 24 weeks of age. Blood glucose levels were higher in both male and female Akita-LDLr −/− mice (137% and 70%, resp.). Male Akita-LDLr −/− mice had markedly increased plasma cholesterol and triglyceride levels, a three-fold increase in atherosclerosis, and enhanced accumulation of macrophages and T-cells in plaques. In contrast, female Akita-LDLr −/− mice demonstrated a modest 29% increase in plasma cholesterol and no significant increase in triglycerides, atherosclerosis, or inflammatory cells in lesions. Male Akita-LDLr −/− mice had increased levels of plasma IL-1β compared to nondiabetic mice, whereas no such difference was seen between female diabetic and nondiabetic mice. Conclusion. Akita-LDLr −/− mice display considerable gender differences in the development of diabetic atherosclerosis. In addition, the increased atherosclerosis in male Akita-LDLr −/− mice is associated with an increase in inflammatory cells in lesions.

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"Immune responses against aldehyde-modified laminin accelerate atherosclerosis in Apoe (-/-) AbstractBackground: LDL oxidation in the vascular wall is associated with aldehyde

Immunization with MDA-fibronectin significantly reduces the development of atherosclerosis in apoE-deficient mice suggesting that the immune response observed in humans may have a protective effect. MDA-fibronectin represents a possible novel target for immunomodulatory therapy in atherosclerosis.